Chromatin Accessibility Analysis Of Piglet Small Intestinal Epithelial Cells Infected With Enterotoxigenic Escherichia Coli F4ac

Enterotoxigenic Escherichia coli(ETEC)F4ac is the main pathogenic bacterium causing diarrhea in newborn and weaned piglets.After ETEC F4 ac in the environment or feed enters the piglet’s intestine,the fimbriae bind to the piglet small intestinal epithelial cell-specific receptor and release a large amount of enterotoxin.This leads to overproduction of proinflammatory factors and destruction of the intestinal barrier,thereby promoting apoptosis of intestinal epithelial cells,thereby accelerating the pathogenesis of diarrhea in ETEC F4 ac piglets.Transcription factors play a key role in immune regulation.Exploring the immune regulatory mechanism of ETEC F4 ac infected hosts,and mining core transcription factors is the key to the breeding of ETEC F4 ac piglets with diarrhea disease resistance.In this study,the immune damage model of ETEC F4 ac infection with small intestinal epithelial cells(IPEC-J2)was first established by different time gradient attacks on porcine IPEC-J2.Then,ATAC-seq and RNA-seq high-throughput sequencing technologies were used to analyze the chromatin accessibility and gene expression changes of IPEC-J2 cells caused by ETEC F4 ac infection,and the core transcription factors and their immune regulatory pathways were screened.Finally,the function of the screened core transcription factor KLF9 was verified to explore its mechanism of action in ETEC F4 ac infected IPEC-J2 cells.The specific results are as follows:(1)In vitro attack(concentration 100:1)was performed on IPEC-J2 cells using different time gradients(3 h,4 h and 5 h),and CCK-8 and flow cytometry detected the changes of cell activity before and after invasion.The results showed that IPEC-J2 cell activity decreased with the extension of ETEC F4 ac invasion time.When the infection time was 4 h,the cell activity decreased to 73% of the uninject group,and the apoptosis rate increased from 3.05% to 11.66%.The m RNA expression levels of inflammatory factors and apoptosis-related genes were detected,and the results showed that the m RNA levels of inflammatory factors IL-1A,CXCL2 and TNF,as well as the apoptotic genes PUMA and GADD45 B,were significantly upregulated(P < 0.05).The above results showed that ETEC F4 ac attack would stimulate elevated expression of immune-related genes and promote apoptosis in IPEC-J2 cells.In this study,we successfully constructed a damage model of in vitro infection of IPEC-J2 cells by ETEC F4 ac.(2)Using the above-constructed in vitro bacterial attack model,RNA-seq and ATAC-seq sequencing of IPEC-J2 cells in the inject and uninject groups were sequenced,and their gene expression changes and chromatin accessibility were analyzed.RNA-seq screened a total of217 differential up-regulated and 462 differential down-regulated genes.Functional enrichment analysis revealed GO entries such as apoptosis processes,inflammatory responses,and p38 cascades,as well as apoptosis,NF-κB and MAPK signaling pathways were significantly enriched.The GSEA analysis results also showed that the above pathways were significantly activated after infection with ETEC F4 ac.Further indicating that these pathways are involved in the immune regulation caused by ETEC F4 ac infection.ATAC-seq screened a total of 7314 differential up-regulated and 5785 differential down-regulated peaks,annotated to 4166 and4915 peak-related genes.These differential peak-related genes were significantly enriched into GO entries such as cell cycle,apoptosis process and P38 cascade,as well as signaling pathways such as MAPK,apoptosis,and pathogenic escherichia coli infection.Which were similar to the enrichment results of transcriptome differential genes.It was explained that changes in chromatin accessibility regulate apoptosis caused by ETEC F4 ac infection by regulating the expression changes of the above immune pathways.PPI protein interaction network analysis showed that the MAPK signaling pathway was directly or indirectly related to the above significantly enriched pathways,suggesting that the MAPK signaling pathway plays an important role in the infection of IPEC-J2 cells by ETEC F4 ac.A total of 13 differential transcription factors were screened out by the combined analysis of ATAC-seq and RNA-seq,including 10 core transcription factors that were consistently upregulated after infection with ETEC F4 ac.These transcription factors were all related to cellular immunity,inflammatory response,cell proliferation and apoptosis,among which KLF9 was the upstream regulator of the p38-MAPK signaling pathway.(3)Based on the results of sequencing and bioinformatics analysis,the role of core transcription factor KLF9 in ETEC F4 ac infected IPEC-J2 cells was further verified.The results showed that after ETEC F4 ac infection with IPEC-J2 cells,the expression level of KLF9 was significantly increased(P < 0.05).Compared with the uninterfered group,the activity of IPECJ2 cells was significantly increased and the apoptosis rate was significantly reduced after interference with KLF9(P < 0.05).Using the JASPAR database,GADD45 B,an upstream gene in the p38-MAPAK signaling pathway,may be a target gene for KLF9.Further experimental results showed that interference with KLF9 significantly inhibited the expression of GADD45B(P < 0.05).The activity of the p38-MAPK signaling pathway and the expression of downstream inflammatory genes IL-6,CXCL2 and apoptotic protein Bax in IPEC-J2 cells after infection were significantly downregulated(P < 0.05).Indicating that KLF9 may participate in ETEC by binding to the target gene GADD45 B to regulate the p38-MAPK signaling pathway F4 ac elicited an immune response.In summary,the damage model of IPEC-J2 cells infected by ETEC F4 ac in vitro was constructed.The differentially expressed genes and chromatin accessibility changes of ETEC F4 ac infection with IPEC-J2 cells were analyzed by RNA-seq and ATAC-seq.P38-MAPK and KLF9 were screened out as core pathways and core transcription factors involved in ETEC F4 ac immune injury.RNA interference was used to verify that the transcription factor KLF9 may affect the activity and apoptosis of IPEC-J2 cells infected with ETEC F4 ac through the KLF9-GADD45B-P38-MAPK signaling pathway.The above results provide a theoretical basis for revealing the molecular mechanism of host immune regulation of ETEC F4 ac infection,and then provide a theoretical basis for future disease resistance breeding work.